Expression of Six Chloroplast Genes in <i>Jatropha curcas</i> Callus under Light and Dark Conditions

doi:10.4236/ajps.2011.25077

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American Journal of Plant Sciences, 2011, 2, 650-656

doi:10.4236/ajps.2011.25077 Published Online November 2011 (http://www.SciRP.org/journal/ajps)

Expression of Six Chloroplast Genes in Jatropha

curcas Callus under Light and Dark Conditions

Wilson Thau Lym Yong, Cassandra Sze Yii Chew, Kenneth Francis Rodrigues

Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kina balu, Sabah, Malaysia.

Email: [email protected].m y; [email protected]

Received September 8th, 2011; revised September 30th, 2011; accepted October 30th, 2011.

ABSTRACT

The induction of genes encoded in the open reading frames (ORFs) of chloroplast genomes have been posited to be in-

fluenced by ambient light condition. The current study focused on determining which of the six ORFs, encoding the

genes ycf 1, ycf 2, psbD (photosystem II), rbcl (Rubisco), matK (Maturase K) and rpoC1 (RNA polymerase) were influ-

enced by light. Characterization of gene expression at the whole plant level and callus stage facilitates the identifica-

tion of transcripts which are differentially regulated under these environmental conditions. Specificity of these primers

was tested against genomic DNA and total RNA. Transcripts of six targeted genes were detected in all three replicates

of the green and white callus under light and dark conditions, except for ycf 2 gene in green callus under light. The re-

sult showed that a partial transcript of the gene ycf 2 located on the J. curcas chloroplast genome was not detectable

using reverse transcription PCR. This finding was then validated using quantitative real-time PCR. The gene was sus-

pected to be post-transcriptionally modified. The transcripts of the remaining five ORFs could be detected using quan-

titative real-time PCR. Specific transcripts can be identified for application as biomarkers for selection of callus for

plantlet regeneration.

Keywords: Chloroplast DNA, Gene Expression, Jatropha curcas, Post-Transcriptional Modification

1. Introduction

Jatropha curcas is a dro ught-resistant oil plan t which be-

longs to the family Euphorbiceae and widely distributed

in tropical and subtropical areas, especially in Central

and South America, Africa, India and Southeast Asia.

The development of J. curcas as a high-yielding and effi-

cient new biofuel source is still at a relatively early stage.

Some tactical strategies such as molecular characteriza-

tion of J. curcas will facilitate the development of this

important oilseed crop [1]. The complete chloroplast

DNA (cpDNA) sequence of J. curcas is now available

(Accession number FJ695500), however there is no data

pertaining for the expression of cpDNA open reading

frames (ORFs).

The cultivation of J. curcas encounters challenges such

as disease problems and climate lead to reduced plant

productivity. Abiotic stress resultin g from abnormal light

conditions is one of the major reasons for declin e in plan t

productivity. According to [2], the expression patterns of

different genes need to be identified and studied for up-

regulation or silencing as part of the transgenic approach

to Jatropha improvement. Integration of gene analysis

with gene discovery and modelling of genetic networks

will facilitate a comprehensive understanding of stress

tolerance, permit the development of useful and effective

molecular markers, and identify candidate genes for ge-

netic transformation and engineering [3].

In order to carry out a preliminary study on the diffe-

rential induction of chloroplast genes under different

light conditio ns, a total of six transcripts representing the

genes ycf 1, ycf 2, psbD (photosystem II), rbcl (Rubis-

CO), rpoC 1 (RNA polymerase) and matK (Maturase K)

were targeted. The approach which was undertaken in-

volved the design and application of primers to target

specific transcripts which have been conjectured to be in-

fluenced by light and dark conditions. The main objec-

tives for this study were to identify the chloroplast genes

which are induced under conditions of light and com-

plete darkness in J. curcas in vivo and in vitro and to

validate expression of genes using real-time PCR.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Two week old of green and white callus cultured under

Expression of Six Chloroplast Genes in Jatropha Curcas Callus under Light and Dark Conditions651

the following conditions: 1) green callus under light and

2) 24 h darkness, 3) white callus under light and 4) 24 h

darkness were used as experimental materials. Three re-

plicates of each green and white callus for each condi-

tion were maintained aseptically on half strength MS

basal medium [4]. Leaves of J. curcas from the germi-

nated plant grown in a pot were used as control.

2.2. Primer Design

Specific primer pairs to characterize gene expression

were designed using Primer 3 software. For the reverse

transcription PCR, primers were designed based on the

intronless chloroplast DNA genome, which is available

at the GenBank (FJ695500). In addition, another set of

primers were designed for real-time PCR assay. These

primers were designed based on the sequence results ob-

tained after reverse transcription PCR.

2.3. DNA Extraction

DNA was extracted from a fully expanded leaf of J. cur-

cas using the 2% CTAB method as previously described

in [5] with few modifications. PCR was performed on

genomic DNA in order to test for the specificity of pri-

mers.

2.4. RNA Extraction, Reverse Transcription and

PCR Analyses

Total RNA was extracted using the RNeasy Plant Mini

Kit (Qiagen) according to the manufacturer’s protocol.

RNA was reverse transcribed into cDNA using the First

Strand cDNA Synthesis Kit (Fermentas). Reverse trans-

cription PCR amplification with specific primers as

shown in Table 1 was done using standard protocols [6]

and the results were applied to determine chloroplast

gene induction in the callus and control plant. All the

amplifications were carried out in 20 µl reaction volume

containing 50 ng of template DNA, 1.5 mM MgCl2, 1X

PCR buffer (10 mM Tris-HCl, 50 mM KCl), 1 U taq

DNA polymerase (Fermentas), 5 µM of primer, 0.2 mM

of dNTP mix in a thermocycler (Eppendorf, Germany).

The PCR conditions for amplification were 95˚C for 3

min, followed by 30 cycles at 95˚C for 30 s, annealing at

56˚C for (primers KYCF2, YCB, YCFD and PS2D2) and

58˚C for (primers YCF1, YCF2, MATK, RBCL and

RPOC1) for 40 s, respectively, 72˚C for 2 min, with a

final extension at 72˚C for 10 min.

2.5. DNA Sequencing and Data Analyses

PCR products corresponding to the expected sizes were

extracted from the agarose gel using QIAGEN Gel Ex-

traction Kit according to the manufacturer’s protocol.

Approximately 40 ng/µl ex tracted PCR produ cts, with 10

pmole/µl of the specific primer, were sent fo r sequen cing

at 1st Base Malaysia using the BigDye® Terminator v3.1

cycle sequencing kit. Nucleotide sequences were ana-

lyzed and compared with the GenBank database of the

National Center for Biotechnology Information (NCBI)

for the similarity using the Basic Local Alignment

Search Tool (BLASTn) online software [7]. The degree

of similarity was determined in terms of the E value.

2.6. Quantitative Real-Time PCR

The genes which were confirmed to be not transcribed by

the reverse transcription PCR were then verified using

quantitative real-time PCR.

In real-time PCR assay, the YCFD2 primers used for

the amplification of the 188 bp fragment of part of ycf 2

gene were:

Table 1. Specific primers for the PCR amplification of the chloroplast genes.

Primer Name ORFs Primers’ Sequence Ta (˚C) Product (bp)

KYCF2 ycf 2 F: TGCAAAGAATCCTCGACGTG

R: AACTCGACGATCCTGTTTGG 56 ~1600

YCB ycf 2 F: CATGGTTATGGACCCGAATC

R: AAATGCCCGGGAGTTCCTCT 56 ~1500

YCFD ycf 2 F: GTTCATCCTTCGGAACCAGA

R: GACCCCCGAATTTGGAGTAT 56 ~1400

PS2D2 psbD F: GATATTATGGATGATTGGTTACGG

R: GTTTCCACGGGGTAGAACCT 56 ~1000

YCF1 ycf 1 F: ATCGAGCATTTCCCCTTTTT

R: TTTCAAATTCCCGAATGGTC 58 ~850

YCF2 ycf 2 F: AAGAGCCGGGAGCAATTTAT

R: CGATAGGGCCGCATTGGTAA 58 ~930

RBCL rbcl F: GACAACTGTGTGGACCGATG

R: CCAAAGATCTCGGTCAGAGC 58 ~990

RPOC1 rpoC 1 F: CGGATGTCCCTGCATAACTT

R: CTACTGGAGCCGGATGAGAG 58 ~990

MATK matK F: CTGCGCGAAATAGAGGAAAC

R: CCCTTCGCTATTGGATGAAA 58 ~970

Expression of Six Chloroplast Genes in Jatropha Curcas Callus under Light and Dark Conditions

652

F: 5’-GAATTCCATTGGACCCAGAA-3’

R: 5’-GCGAACCCCAGTTAGATTCA-3’

The RPOC1b primers for the 178 bp fragment of the

rpoC 1 gene were:

F: 5’-CCCGAGTTGAGACCAATCAT-3’

R: 5’-CCACGGCTTCTTGTACCAAT-3’.

Gene expression was analyzed by real time quantita-

tive PCR using the SYBR Green system in an iQ5 Real-

Time PCR detection system (Bio-Rad). Real-time PCR

was carried out in 50 µl reaction volume which consisted

25 µl iQ SYBR Green Supermix (Bio-Rad), 1 µl of 0.5

µM of each forward and reverse primers, 3 µl of RNA

and 20 µl of nuclease free water. PCR reactions were

performed under the following thermal cycling condi-

tions: 1 min at 50˚C, 5 min at 95˚C, 50 cycles of 10 s at

95˚C and 30 s at 58˚C. Results were confirmed by re-

solving the product on a 1.5% agarose gel.

3. Results and Discussion

3.1. DNA and RNA Extraction

Using the modified CTAB protocol, the concentration of

DNA obtained from J. curcas leaves ranged from 16 - 30

µg/ml. The A260/A280 ratio was greater than 1.8, indicat-

ing DNA purity. The modified CTAB DNA extraction

method was effective in isolating DNA, which could be

used for testing the specificity of primers using PCR.

The CTAB protocol for DNA extraction worked well for

extracting high quality DNA from leaf samples. The

CTAB protocol is a rapid and technically easy method

for preparing nucleic acids that can be amplified using

PCR. This method has been widely applied on various

highly consistence of secondary metabolites and lipid

plants , s u ch as Targetes minuta L. [8 ], oil p alm [9 ] an d J.

curcas [10].

The RNeasy Plant Mini Kit (Qiagen) was able to ex-

tract high quality RNA from J. curcas callus. The integ-

rity of RNA was judged by the sharpness of ribosomal

RNA bands visualized on 1.5% agarose gel. For all

tested RNA samples, distinct 25S and 18S rRNA bands

were observed in agarose gel. The A260/A230 in the range

1.9 - 2.0 indicated the purity of RNA was suitable for

downstream applications. The high quality of RNA was

isolated from callus using RNeasy Plant Mini Kit

(Qiagen). The RNeasy extraction procedure utilizes a mi-

nimum number of reagents and steps, thus reducing the

handling time and minimizing the risk of RNase con-

tamination.

3.2. PCR Amplification

In this study, four different primers which amplified spe-

cific locus of ycf 2 gene in J. curcas chloroplast genome

(Figure 1) were tested. These transcripts accumulated in

Figure 1. Amplification of ycf 2 gene targeted at different

regions in J. curcas chloroplast genome (FJ695500).

green and white callus under light and darkness when

amplified with YCB, YCF2 and KYCF2 primers. How-

ever, reverse transcription PCR did not detect gene am-

plified by YCFD primer for the green callus under light

treatment and no PCR product was detected. Amplicons

with the product size of 1433 bp were visualized from

the PCR products of green callus and white callus in dark

and white callus under light (Figure 2). It was predicted

that part of the ycf 2 transcript located at position of

94050 to 95483 of J. curcas chloroplast genome (FJ695500)

was not transcribed in green callus under light condition

and it was suspected to be post-transcriptionally modi-

fied. Higher eukaryotes respond to environmental signals

by regulating their genes. Specifically, gene expression is

controlled at two levels. First, transcription is controlled

by limiting the amount of mRNA that is produced from a

particular gene. The second level of control is through

post-transcriptiona l events that regulate the translation of

mRNA into proteins. Even after a protein is translated,

post-translational modifications can affect its activity

[11].

The ycf 1 gene transcripts were found in both green

and white callus in both light and dark conditions. The

results indicated the transcripts from ycf 1 did accumu-

late in all the callus treated with differential light condi-

tions and the ycf 1 gene is suspected to be not related to

photosynthesis. Although the function of the ycf 1 gene

has not been ascertained, its presence in a non-photo-

synthetic plant named Epifagus virginiana and experi-

ments in tobacco indicate that it may have function

which is probably not related to pho tosynthesis [12].

Figure 2. Electrophoresis of PCR products of cDNA ampli-

fied by YCFD primer on a 1.5% agarose gel. Lane M: 1 kb

O’ GeneRuler™ ladder (Fermentas); Lane 1-2: green callus

under light; Lane 3-4: green callus in dark; Lane 5-6: white

callus under light; Lane 7-8: white callus in dark.

Expression of Six Chloroplast Genes in Jatropha Curcas Callus under Light and Dark Conditions653

PCR amplification of the rpoC1 gene yielded 945 bp

fragments for all three replicates of each treatment of

callus cDNA and leaf under daily natural photoperiod.

As shown in Figure 3, the cDNA under all treatments

mentioned were successfully amplified by the reference

gene. This reference gene, encoded the β’ subunit of

RNA polymerase, was expressed in the treated callus and

leaf at all times. The rpoB and rpoC genes are house-

keeping genes that can be utilized in plant transcripto-

mics [13]. The psbD gene, which is a component of pho-

tosystem II and responsible for photosynthetic, is not

affected by the light and dark treatment in this study.

Both the green and white callus treated under light and

dark were amplified by the psbD specific gene primers

(PS2D2 primers). The matK was chosen as its region is

universally present in land plants and only few excep-

tions of a secondary loss or reorganisations are known to

date [14]. In add ition, th e rbcl gene was chosen as one of

the reference gene due to its characteristic of multicopy

chloroplast sequence an d it is highly conserved sequence

in plants [15].

3.3. Nucleotide Sequence Alignments and

Database Searches

To further confirm the presence of specific chloroplast

genes in callus, the amplified products were sent for nu-

cleotide sequencing. The respective sequences obtained

were assembled using DNASTAR SeqManII™ software

and then compared with the GenBank database of the

NCBI using the BLASTn software. From BLASTn, the

sequences obtained from the amplification of genomic

DNA leaf and the amplification from cDNA callus were

compared. Results showed the sequences achieved high

score and expectation (E) value near zero when com-

pared against the J. curcas chloroplast genome (FJ695500).

These data indicated the sequences resulted from se-

quencing had high similarity and there was no discern-

able polymorphism in these genes.

3.4. Real-Time PCR

The regulation of ycf 2 gene transcripts was validated

using the quantitative real-time PCR. From the amplifi-

cation plot shown in Figure 4(a), the red plot in the am-

plification curve indicated there was no amplification for

green callus under light. The result was confirmed to be

similar to the reverse transcription PCR, whereas the

gene was not transcribed in the green callus under light.

To further verify the ycf 2 gene amplified by YCFD pri-

mers, real-time PCR was carried out to detect the part of

ycf 2 chloroplast gene induction under differential light

condition. In the real-time assay, only YCFD primers

and RPOC1 primers were performed on the callus cDNA.

They were chosen due to the validation of ycf 2 gene

Figure 3. Electrophoresis of PCR products of cDNA ampli-

fied by RPOC1 reference primer on a 1.5% agarose gel.

Lane M: 1 kb O’ GeneRuler™ ladder (Fermentas); Lane 1- 3:

green callus under light; Lane 4-6: green callus in dark;

Lane 7-9: white callus in dark; Lane 10: leaf (control);

Lane 11: negative control.

primed by YCFD while rpoC 1 was chosen as the house-

keeping gene.

As mentioned earlier, failure of transcript detection in

part of gene may due to post-transcriptional modification.

The chloroplast genome differs in many respects from

the nuclear genome. While chloroplast genes are regul-

ated at the transcriptional level, po st-transcriptional gene

regulation is predominant. As might be expected, light

plays an important role in the regulation of many chlo-

roplast genes. According to [16], RNA processing, de-

gradation, and translation are the predominant levels of

gene regulation in the chloroplast genome. Post-trans-

criptional gene regulation is a complex, multistage phe-

nomenon that helps to fine tune the levels of active pro-

teins in the cell.

Primary translation products often undergo a variety of

modification reactions, involving the addition of chemi-

cal groups which are attached covalently to the polypep-

tide chain at the translation al and post-translational levels.

Study carried out by [17] stated plants’ subsistence de-

pends on their ability to rapidly regu late gene expression

in order to adapt their physiology to their environment.

Post-transcriptional regulation of gene expression plays

an important role in how plants respond to abiotic stresses

such as light inten sity, te mperature an d the av ailab ility of

water and essential nutrients. In addition, [18] also ca-

rried out a study on chloroplast ycf 2 gene expressions in

stressed plants. The study focused on the fruit develop-

ment in order to verify if ycf 2 may have a specialized

function in non-photosynthetic tissue during seed matu-

ration.

The candidate reference gene rpoC 1 was used to am-

plify against the cDNA. Figure 5(a) demonstrated the

efficiency and sensitivity in the amplification of refe-

rence gene. Five steep curves were observed in the am-

plification profile and this suggested that the reference

gene was expressed in all samples under different treat-

ments including the leaf. In addition , in order to confirm

the presence of the specific PCR product with product

size of approximately 170 bp, the real-time PCR pro-

Expression of Six Chloroplast Genes in Jatropha Curcas Callus under Light and Dark Conditions

654

Figure 4. Real-time assay. (a) Real-time PCR amplification plot of cDNA from four treatments of J. curcas callus amplified

by YCFD2 primer using SYBR Green dye. (b) Electrophoresis of real-time PCR products with approximately 180 bp on

1.5% agarose gel. cDNA was used as template and amplified against YCFD. Lane 1: green callus under light; Lane 2: white

callus under light; Lane 3: green callus in dark, Lane 4: white callus in dark; Lane 5: leaf; Lane M: 100 bp ladder (Promega).

Figure 5. Real-time assay. (a) Real-time PCR amplification plot of cDNA from four treatments of J. curcas callus amplified

by reference primer RPOC1b, using SYBR green dye. (b) Electrophoresis of real-time PCR products with approximately 170

bp on 1.5% agarose gel. cDNA was used as template and amplified against candidate reference gene rpoC 1. Lane M: 100 bp

ladder (Promega); Lane 1: green callus under light; Lane 2: white callus under light; Lane 3: green callus in dark; Lane 4:

white callus in dark; Lane 5: leaf.

ducts were resolved on 1.5% agarose gel (Figure 5(b)).

Under the PCR conditions tested, there was amplification

in all the cDNA under four different treatments and leaf

against the reference gene rpoC 1.

As illustrated in the melting curve in Figure 6, small

variations in the Tm indicated a different pattern of am-

plification. For Figure 6(a), there were four peaks (each

represented by green callus under dark, white callus un-

der light and dark, and leaf) presented in the melting

curve with Tm of 82.5˚C. Five peaks (each represented

by green callus under light and dark, white callus under

light and dark, and leaf) with Tm in the range of 80˚C to

81˚C, were apparent in the melting curve shown in Fig-

ure 6(b). Other than the significant single sharp peak,

there was an extra single low peak produced. This low

peak was suspected to be generated by non-specific am-

plification.

4. Conclusion s

Information pertaining to the abiotic stress respon se of J.

curcas, with specific reference to light stress will provide

a basis for selection of callus tissue for regeneration in

plant tissue culture systems. This investigation provided

an insight into the expression of the ycf 2 gene and estab-

Expression of Six Chloroplast Genes in Jatropha Curcas Callus under Light and Dark Conditions655

Figure 6. Melting curve profile for real-time PCR amplification of cDNA from four treatments of J. curcas callus. (a) cDNA

primed with YCFD2. (b) cDNA primed with RPOC1b.

lished a foundation fo r further analysis of this little stud-

ied gene.

5. Acknowledgements

The authors wish to thank the Biotechnology Research

Institute, Universiti Malaysia Sabah for funding the re-

search and providing facilities for the research to be car-

ried out.

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